The underlying mechanisms of the progressive nature of emphysema remain unclear. Inflammation alone cannot fully explain how emphysema progresses, especially in the late phase. Our previous award revealed new important mechanisms leading to the two hypotheses to be tested in this proposal. The first hypothesis is: Independent of how emphysema is initially triggered, its progression in the late phase results primarily from mechanical force-induced breakdown of the lung parenchyma during breathing. Our second hypothesis is: Mechanical forces start to contribute to the progression of emphysema when the amount of newly synthesized collagen deposited in the alveolar wall relative to elastin reaches a critical threshold. To test these hypotheses, we propose to investigate a control group and the following mouse models of emphysema: a) an elastase treated group which involves inflammation followed by protease/antiprotease imbalance, b) tight skin mouse with abnormal matrix assembly, and c) mice with chronic over-expression of collagenase without inflammation. Using several novel techniques, we will evaluate the mechanical properties of the in vivo whole lungs, isolated tissue strips, alveolar walls and collagen fibers as well as the heterogeneity of the parenchymal structure at three time points during the progression of emphysema. We anticipate that these properties may not be similar in the early stages, but will converge during the late phase of emphysema. To test how collagen assembly affects the failure properties of the lung tissue, we will use a unique Red Fluorescent Protein-collagen that can be used in conjunction with two-photon second harmonic generation microscopy to visualize both the newly synthesized and the existing old collagen during failure tests. Additionally, we will assess the contents of type I and III collagen, elastin and several small molecules such as proteoglycans that are known to influence collagen assembly. The proposed work will a) identify the biophysical conditions that must occur for the dominant cause of tissue destruction in emphysema to be breathing-induced mechanical forces, b) establish links between these biophysical conditions and macroscopic measures of structure and function, and c) identify a threshold beyond which emphysema becomes irreversible. These results will motivate a more rational approach to detection, treatment design, and treatment assessment of emphysema.